Transcriptomics of a Greenlandic Snailfish Reveals Exceptionally High Expression of Antifreeze Protein Transcripts.

Polar fishes have evolved antifreeze proteins (AFPs) that allow them to survive in subzero temperatures. We performed deep transcriptomic sequencing on a postlarval/juvenile variegated snailfish, Liparis gibbus (Actinopterygii: Scorpaeniformes: Cottoidei: Liparidae), living in an iceberg habitat (-2°C) in Eastern Greenland and report detection of highly expressed transcripts that code for putative AFPs from 2 gene families, Type I and LS-12-like proteins (putative Type IV AFPs). The transcripts encoding both proteins have expression levels among the top <1% of expressed genes in the fish. The Type I AFP sequence is different from a reported Type I AFP from the same species, possibly expressed from a different genetic locus. While prior findings from related adult sculpins suggest that LS-12-like/Type IV AFPs may not have a role in antifreeze protection, our finding of very high relative gene expression of the LS-12-like gene suggests that highly active transcription of the gene is important to the fish in the iceberg habitat and raises the possibility that weak or combinatorial antifreeze activity could be beneficial. These findings highlight the physiological importance of antifreeze proteins to the survival of fishes living in polar habitats.

Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill.

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7's enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

A putative chordate luciferase from a cosmopolitan tunicate indicates convergent bioluminescence evolution across phyla.

Pyrosomes are tunicates in the phylum Chordata, which also contains vertebrates. Their gigantic blooms play important ecological and biogeochemical roles in oceans. Pyrosoma, meaning "fire-body", derives from their brilliant bioluminescence. The biochemistry of this light production is unknown, but has been hypothesized to be bacterial in origin. We found that mixing coelenterazine-a eukaryote-specific luciferin-with Pyrosoma atlanticum homogenate produced light. To identify the bioluminescent machinery, we sequenced P. atlanticum transcriptomes and found a sequence match to a cnidarian luciferase (RLuc). We expressed this novel luciferase (PyroLuc) and, combined with coelenterazine, it produced light. A similar gene was recently predicted from a bioluminescent brittle star, indicating that RLuc-like luciferases may have evolved convergently from homologous dehalogenases across phyla (Cnidaria, Echinodermata, and Chordata). This report indicates that a widespread gene may be able to functionally converge, resulting in bioluminescence across animal phyla, and describes and characterizes the first putative chordate luciferase.

Disrupting Fluorescence by Mutagenesis in a Green Fluorescent Fatty Acid Binding Protein from a Marine Eel.

Biofluorescence has been found to be an increasingly widespread phenomenon in the ocean. The reclusive Caribbean chlopsid eel, Kaupichthys hyoproroides displays bright green fluorescence in its native marine environment. We have previously shown the fluorescence to be attributed to a fluorescent fatty acid-binding protein, Chlopsid FP, part of a larger family of fluorescent fatty acid-binding proteins, including the homologous UnaG. All require the addition of exogenous bilirubin for fluorescence. Here, we report the generation of a series of point mutants, and deletions that result in the quenching of fluorescence in Chlopsid FP. In addition, we report the binding constants of bilirubin to Chlopsid FP and mutants, measured by fluorescence titration. This study provides key insights into the potential mechanism of fluorescence in this class of fluorescent fatty acid-binding proteins.

Bright Green Biofluorescence in Sharks Derives from Bromo-Kynurenine Metabolism.

Although in recent years there has been an increased awareness of the widespread nature of biofluorescence in the marine environment, the diversity of the molecules responsible for this luminescent phenotype has been mostly limited to green fluorescent proteins (GFPs), GFP-like proteins, and fluorescent fatty acid-binding proteins (FABPs). In the present study, we describe a previously undescribed group of brominated tryptophan-kynurenine small molecule metabolites responsible for the green biofluorescence in two species of sharks and provide their structural, antimicrobial, and spectral characterization. Multi-scale fluorescence microscopy studies guided the discovery of metabolites that were differentially produced in fluorescent versus non-fluorescent skin, as well as the species-specific structural details of their unusual light-guiding denticles. Overall, this study provides the detailed description of a family of small molecules responsible for marine biofluorescence and opens new questions related to their roles in central nervous system signaling, resilience to microbial infections, and photoprotection.

Luciferin production and luciferase transcription in the bioluminescent copepod Metridia lucens.

Bioluminescent copepods are often the most abundant marine zooplankton and play critical roles in oceanic food webs. Metridia copepods exhibit particularly bright bioluminescence, and the molecular basis of their light production has just recently begun to be explored. Here we add to this body of work by transcriptomically profiling Metridia lucens, a common species found in temperate, northern, and southern latitudes. In this previously molecularly-uncharacterized species, we find the typical luciferase paralog gene set found in Metridia. More surprisingly, we recover noteworthy putative luciferase sequences that had not been described from Metridia species, indicating that bioluminescence produced by these copepods may be more complex than previously known. This includes another copepod luciferase, as well as one from a shrimp. Furthermore, feeding experiments using mass spectrometry and 13C labelled L-tyrosine and L-phenylalanine firmly establish that M. lucens produces its own coelenterazine luciferin rather than acquiring it through diet. This coelenterazine synthesis has only been directly confirmed in one other copepod species.

Biofluorescence in Catsharks (Scyliorhinidae): Fundamental Description and Relevance for Elasmobranch Visual Ecology.

Biofluorescence has recently been found to be widespread in marine fishes, including sharks. Catsharks, such as the Swell Shark (Cephaloscyllium ventriosum) from the eastern Pacific and the Chain Catshark (Scyliorhinus retifer) from the western Atlantic, are known to exhibit bright green fluorescence. We examined the spectral sensitivity and visual characteristics of these reclusive sharks, while also considering the fluorescent properties of their skin. Spectral absorbance of the photoreceptor cells in these sharks revealed the presence of a single visual pigment in each species. Cephaloscyllium ventriosum exhibited a maximum absorbance of 484 ± 3 nm and an absorbance range at half maximum (λ1/2max) of 440-540 nm, whereas for S. retifer maximum absorbance was 488 ± 3 nm with the same absorbance range. Using the photoreceptor properties derived here, a "shark eye" camera was designed and developed that yielded contrast information on areas where fluorescence is anatomically distributed on the shark, as seen from other sharks' eyes of these two species. Phylogenetic investigations indicate that biofluorescence has evolved at least three times in cartilaginous fishes. The repeated evolution of biofluorescence in elasmobranchs, coupled with a visual adaptation to detect it; and evidence that biofluorescence creates greater luminosity contrast with the surrounding background, highlights the potential importance of biofluorescence in elasmobranch behavior and biology.

Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment.

We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.

Titanium mineralization in ferritin: a room temperature nonphotochemical preparation and biophysical characterization.

The incremental addition of titanium(III) citrate to H-chain homopolymers of human ferritin results in the formation of 1.5-6.5-nm particles of amorphous TiO(2) within the nanocage of the protein. The mineralization conditions are mild, featuring ambient temperature and no need for photochemical activation. Low ratios of titanium to protein favor intraprotein mineralization, and the products are characterized by stained and unstained transmission electron microscopy, UV-vis spectroscopy, dynamic light scattering, analytical ultracentrifugation, and metal analysis. With up to 1,000 equiv of metal, there is no change to the protein hydrodynamic radius or diffusion constant. There is, however, a systematic shift in the sedimentation coefficient, which confirms mineralization within the protein core.

Beyond bilobal: transferrin homologs having unusual domain architectures.

BACKGROUND: Most transferrin family proteins have a familiar bilobal structure, the result of an ancient gene duplication, with an iron binding site in each of two homologous lobes. Scattered throughout the evolutionary tree from algae to mammals, though, are transferrin homologs having other kinds of domain architectures. SCOPE OF REVIEW: This review covers a variety of unusual transferrin forms, including monolobals, bilobals with one or both iron-binding sites abrogated, bilobals accessorized with long insertions or with membrane anchors, and even trilobals. The monolobal transferrin homologs from marine invertebrate ascidians are especially highlighted here. MAJOR CONCLUSIONS: Unusual transferrin homologs appear scattered through much of the evolutionary tree. For some of these proteins, iron binding and/or iron transport appear to be the primary roles; for others they clearly are not. Many are incompletely or not at all studied. GENERAL SIGNIFICANCE: Taken together, these proteins begin to offer a glimpse into how the transferrin architecture has been repurposed for a diversity of applications. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

The challenges of trafficking hydrolysis prone metals and ascidians as an archetype.

Some of the metal ions that are required, exploited, or simply managed in biological systems are susceptible to hydrolysis and to hydrolytic precipitation in the aqueous, aerobic environment of much of biology. Organisms have evolved exquisite mechanisms for handling these metal ions, offering striking examples of biological control over inorganic coordination chemistry. This year marks the one hundredth anniversary of the discovery of remarkably high vanadium concentrations in the blood cells of the ascidian. In the ensuing years, these marine invertebrates were established as masters of the biological chemistry of very hydrolysis-prone metals, with various ascidian species accumulating high concentrations of iron, vanadium, and titanium, among others. These three metals have very different histories of biological relevance, and many questions remain about how, and ultimately why, these organisms sequester them. This Perspective addresses the aqueous coordination chemistry that organisms like ascidians must control if they are to manipulate hydrolysis-prone metal ions, and describes some of the ascidian biomolecules that have been implicated in this phenomenon. The recently available genome sequence for one ascidian species offers a glimpse into its metal-management arsenal. It offers the opportunity to map the relatively well-studied paradigm of iron management onto the genome of an organism that is intermediate in evolution between invertebrates and vertebrates. The ascidians have much to teach us about how to manage metals like iron, titanium, and vanadium and how that ability evolved.

Contrasting synergistic anion effects in vanadium(V) binding to nicatransferrin versus human serum transferrin.

Some ascidians sequester vanadium and other metal ions that are bound and transported in higher organisms by transferrin. The ascidian Ciona intestinalis has a monolobal transferrin (nicatransferrin) in its plasma. The binding of vanadium(V) to nicatransferrin was investigated by using isothermal titration calorimetry and UV-vis spectroscopy, in the presence and absence of NaHCO(3), and was compared with human serum transferrin. Nicatransferrin and serum transferrin bind V(V) with similar strengths [K = (2.0 +/- 0.6) x 10(5) M(-1) for nicatransferrin]; however, nicatransferrin requires a synergistic anion for V(V) binding, whereas serum transferrin does not. Spectroscopy supports a different kind of coordination site.

Enhanced conductivity of thin film polyaniline by self-assembled transition metal complexes.

In a recent study, the transition metal complex, cis-dichlorobis(2-,2'-dipyridyl)ruthenium (II) (Ru(bpy)2Cl2), and the macrocycle Ru(TPP)CO (TPP:- tetraphenylporphine) were bound to pyridine terminated self-assembled monolayers on quartz. Following modification of the quartz surface with metal complexes, the conducting polymer polyaniline was deposited via in situ polymerization. The sheet conductivity (as measured by the four-probe method) of the resulting polyaniline films deposited onto Ru(bpy)2Cl2 and Ru(TPP)CO surfaces was significantly enhanced relative to films deposited onto unmodified quartz. It is postulated that either the macrocycle or the transition metal complex-modified surface interacts with the conducting polymer as it is forming, resulting in a more ordered expanded coil conformation for the polymer. The net result of such an interaction is a thin film possessing significantly greater electrical conductivity.